Microbial Diversity, Methanogen Isolation and Methane Producing Potenals in Methane Hydrate Regions of Sw Offshore Taiwan

甲烷水合物廣泛分佈於極區永凍層及陸緣海域等處。全球甲烷水合物的甲烷蘊藏量，保守估計至少有20 × 1015 立方公尺／一大氣壓，約為目前已知全球化石燃料等能源資源之有機碳總儲量的兩倍。相較於傳統的化石燃料，甲烷水合物是較為潔淨的能源，極可能成為二十一世紀最重要能源資源之一。目前一般認為甲烷的形成主要源自微生物作用或熱分解作用二種。由美國東南、北加州、祕魯、墨西哥灣等遠濱海域地區所採回甲烷水合物及含甲烷水合物的沉積物巖樣之烷氫類氣體組成及甲烷中碳同位素δ13C的分析結果顯示，絕大部分的甲烷水合物所含之甲烷係源自微生物作用。微生物中能生產甲烷氣體的僅有甲烷古菌(methanogen，甲烷菌，甲烷太古生物)。在大自然中，絕對厭氧的甲烷古菌能在任何無氧、低氧化還原電位的環境，利用簡單的H2+CO2, formate, acetate, methanol及methylamines作為主要碳源，進行甲烷化作用(methanogenesis)產生甲烷，提供能源和大氣中碳的循環並在生物的食物鏈中佔有相當基礎且重要的地位。產生甲烷氣體的甲烷化作用(methanogenesis)是甲烷古菌獨特且唯一獲得能量的特殊無氧呼吸方式。甲烷化作用相當複雜，反應所需的酵素系統、coenzyme及調控系統等所參與的基因佔其全部基因的25 % 左右。絕對厭氧的甲烷古菌能在任何無氧、低氧化還原電位的環境生長，對溫度、鹽度和水壓有相當高的極端適應性。目前已經純化的甲烷古菌中，能生長的溫度範圍由2-110 ℃，鹽濃度 0-4.5 M NaCl且能承受1- 600 atm或更高的水壓。 我們實驗室用來探討高鹽滲透逆境適應的嗜鹽甲烷古菌 (Methanohalophilus)能生長在1.2-4.3 M NaCl的環境，也自本土自然環境如彰化王功二林溪附近海域及養殖區、淡水河域、墾丁海域及車籠埔斷層地底取樣，純化甲烷古菌。 目前已純化約30株甲烷古菌，包含四株新種Methanofollis aquaemaris、Methanofollis formosanus、Methanocalculus taiwanensis及Methanocalculus chunghsingensis。甲烷水合物和甲烷古菌(Methanogen)的關係及甲烷水合物蘊藏區域的微生物生態結構在美國與日本等都有團隊進行探討。以16S rDNA基因的分析方式探討甲烷水合物沉積物附近微生物分佈發現微生物的種類相當多樣化，但太古生物(Archaea)的多樣性則相對侷限，主要以硫還原古菌、厭氧甲烷氧化古菌(AOM)與甲烷古菌為主。近一步分析顯示，甲烷水合物之間及上層，硫還原古菌與厭氧甲烷氧化古菌量較高，因此不少學者認為此類微生物族群架構可能影響甲烷水合物的穩定性。甲烷古菌則於甲烷水合物區域下方及附近的底泥占較高的比例。但不同樣區與不同方法的分析結果仍有些矛盾之處，因此甲烷自何處來仍有爭議。不過目前全球各樣區以16S rDNA或rRNA所分析的環境核酸樣品序列顯示，甲烷水合物區域的甲烷古菌多以利用H2+CO2為基質的 Methanomicrobiales與acetate為基質的Methanosarcinales為主。但是，這些研究大多尚未將微生物純化出來，目前唯一自甲烷水合物區域純化出的甲烷古菌是David R. Boone實驗室與日本科學家合作自日本Nankai trough取得的一株利用H2+CO2 和 formate為基質的中溫球菌Methanoculleus submarinus。台灣學界曾在臺灣西南海域發現有天然氣水合物廣泛分佈於南海大陸坡及南臺灣增積巖體中的證據。此研究目的是探討臺灣西南海域天然氣水合物沉積物的微生物生態結構與純化甲烷古菌，藉由我們實驗室的甲烷古菌純化技術純化此類特殊環境中的甲烷古菌，分析其特性以瞭解其特殊甲烷生合成與累積甲烷水合物的機制。另藉由16S rDNA基因轉殖定序與系統演化分析比對及即時定量PCR的應用，以分析甲烷水合物環境微生物的種類與量，進而構築其代謝關係以瞭解甲烷水合物微生物生態圈的功能。Methane hydrates are an ice-like material in which methane molecules are trapped within cages of the crystalline lattice of water molecules. They are distributed along coastal margins, in the sediment of active and passive continental slope margins, as well as in terrestrial regions, trapping enormous volumes of methane, estimated at about twice the amount of all other known fossil fuel reserves. They have attracted much attention as an alternative energy resource potential, and may impact the global environment through generating slope instability.With most of the methane trapped in known hydrate formations being of biogenic origin, this represents a significant new source of natural gas from biological methanogenesis. Methanogenic archaea (Methanogen) are strict anaerobes that share a complex biochemistry for methane synthesis as part of their anaerobic respiration for energy metabolism. Genes involved in this unique methane producing process occupy more than 25% of coding genes of methanogen. Moreover, methanogens are the only Archaea that are truly cosmopolitan. Methanogenic species have been isolated from virtually every habitat in which anaerobic biodegradation of organic compounds occurs, moreover, isolates have also been obtained from geothermal springs, deep-sea vents and hypersaline environments. We have purified 30 strains of methanogens from Taiwan. Among them, four strains have been identified and characterized as new species and they are Methanofollis aquaemaris, Methanocalculus taiwanensis, Methanofollis formosanus and Methanocalculus chunghsingensis. Lately, we have isolated and characterized two novel methanogenic isolates from deep subsurface environment at the depth of 2000 m below land surface (mbls) of Chelungpu and San-Yi fault zones at Dai-Keng, Taichung. One is formate utilizing Methanobacterium palustre and the other is methanol utilizing Methanolobus sp..Microbial numbers are high in hydrate-bearing regions and metabolic studies of these sediments, based on incubations with stable isotopes of substrate and subsequent measurement of the isotope-containing product, have demonstrated methanogenesis, sulfate reduction, and methane oxidation. Phylogenetic analysis of DNA extracted from marine sediments in a methane hydrate zones have been investigated in Gulf of Mexico, Cascadia Margins, Nankai Trough and many elsewhere, which revealed similar but diverse results. In general, they all showed high diversity of bacteria and low diversity of Archaea. The sulfate reducers and anaerobic methane oxidization archaea often occur in the overlying and interior layers of hydrate. Methanogenic archaea including members of the orders Methanobacteriales and Methanosarcinales are often detected more abundantly at sediments below or nearby the hydrate. However, with tremendous environmental 16S DNA clone library, there is only one methanogen - Methanoculleus submarinus related with gas hydrate area has been isolated and characterized.The research team from Taiwan, supported by the Central Geological Survey (CGS) of the Ministry of Economic Affairs (MOEA), has been demonstrated lately that there are potential gas hydrate regions at SW offshore Taiwan. In this research proposal, we plan to investigate the microbial community structure of hydrate regions by phylogenetic analysis of 16S rDNA and mcr gene library. The microbial community analysis will provide the information of microbial ecological interaction to maintain the stability of the methane hydrate zones. In addition, we would like to enrich, isolate and character the methanogens from the hydrate area and use these isolates to study their methane producing capability and mechanism of hydrate formation

Application Potential of Archaeal Compatible Solutes in Food and Agriculture

相容質與滲透壓的基礎與應用研究，從抗鹽相容質glycerol、trehalose和近年發展的高鹽細菌生產的ectoine在發酵工業、食品界、化妝美容界、製藥與醫療等的應用，可以想見微生物因應鹽與滲透壓力的小分子相容質在基礎研究與應用的重要性。 極端厭氧的甲烷太古生物 (Archaea, 又稱古菌、古生菌或古細菌)，以甲烷的生合成方式提供細胞所需的能量，能生存於各式溫度(2- 117 ℃)、酸鹼度(中性至強鹼)及鹽度(淡水至飽合鹽度)等環境，是唯一能適應多變化環境的太古生物。 我們實驗室收集國內外能生長在各種不同鹽度的甲烷菌，長期探討甲烷菌對鹽逆境的反應與適應機制。我們的研究顯示嗜鹽甲烷菌具有一高親和性、高專一性，需能且受滲透壓調控的ABC型式的甜菜鹼運輸系統Bta，會自胞外環境優先攝取相容質甜菜鹼。而當胞外環境缺乏相容質的提供時，能自體生合成相容質β-glutamine、Nε-acetyl-β-lysine及甜菜鹼 (betaine)於細胞體內，以維持膨壓及保護胞內蛋白等大分子。某些相容質具有特殊風味，廣泛應用於食品添加工業上，在農漁業上的應用逐漸受到矚目，已有研究指出帶有相容質基因的轉殖株對於高鹽、低溫、乾旱等環境因子具有耐受性。另外在飼料中添加相容質對畜牧業也極有幫助。絕對厭氧的甲烷太古生物能生長在淡水至飽和鹽環境，對鹽逆境與滲透壓逆境的適應是生物界中最寬廣的，期由對甲烷太古生物在鹽逆境的反應與適應機制相關基因與調控的了解及應用潛力的分析，進而提高生物抗鹽抗旱的能力。我們已獲得嗜鹽甲烷菌Methanohalophilus portucalensis FDF1相容質betaine的生合成酵素GSMT與SDMT的基因。在甲烷菌的共通相容質Nε-acetyl-β-lysine生合成基因部分，我們已分別獲得海洋甲烷菌Methanosarcina mazei N2M9705、耐鹽甲烷菌Methanocalculus chunghsingensis K1F9705b與嗜鹽甲烷菌Methanohalophilus portucalensis FDF1的lysine 2,3-aminomutase (ablA)與β-lysine acetyltransferase (ablB)基因；以及淡水型甲烷菌M. mazei N2M9705的相容質β-glutamine 的生合成基因。在此三年期計畫期間，除了探討甲烷菌自體生合成相容質基因對於環境逆境的調控機制以及分析異源表現蛋白活性之外，並將生合成相容質β-glutamine、Nε-acetyl-β-lysine及betaine的基因組，分別轉殖到大腸桿菌、阿拉伯芥與斑馬魚上，分析其相容質生成累積量與探討是否能提高其對鹽度的耐受性，增加轉殖動、植物對於環境逆境的適應性。將更近一步測試並量產相容質，評估在食品工業上與農林畜牧與養殖業的應用。Archaea as the strict anaerobic, low redox required methanogen, hyperthermophiles that can grow up to 121 ℃, microbes that enjoy the 1000 atm pressure and extreme halophiles that could grow at saturated NaCl. As we facing the extreme living environmental challenge now and in near future, the extreme adaptation genes, extremozymes and strategies from Archaea may help us to fight for the global warming.The osmotic strength of the environment is one of the parameters that determine the ability of organisms to proliferate in a given habitat. Changes in the extracellular osmolarity have the same physiochemical effects on cell from all living domains and the responses to osmotic shifts have considerable similarities in all organisms. Strict anaerobic methanogenic archaea can survive at broad range of salt stress (0-4.5 M NaCl) from freshwater, marine to hypersaline environments. The extreme and board range of salt adaptations made the Methanoarchaea the best model for studying salt-stress response of archaea and osmoadaptation. To encounter the changing osmotic stress in hypersaline environments, halophilic methanogen-Methanohalophilus portucalensis could transport compatible solute (osmolyte) betaine through an energy-requiring, high-affinity and highly specific betaine transport system which was regulated by betaine and osmotic stresses. Without the extracellular supply of betaine, cell could de novo synthesize betaine, Nε-acetyl-β-lysine, β-glutamate and β-glutamine as compatible solutes to increase the internal osmolarity and protect cellular macromolecules.In order to reveal how methanogen deal with salt stress with osmolytes, we have already obtained the (1).osmolyte betaine synthesizing genes for GSMT and SDMT from halophilic methanogen- M. portucalensis; (2). osmolyte Nε-acetyl-β-lysine synthesizing genes encoding for lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB) from marine Methanosarcina mazei strain N2M9705, halotolerant Methanocalculus chungshingensis and halophilic M. portucalensis and (3). osmolyte β-glutamine synthesizing genes encoding for glutamine synthetase (glnA1) from Methanosarcina mazei strain N2M9705.In this grant proposal, we plan to (1) transform osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately to osmolyte transport and synthesis defective mutant E. coli KH13(ΔbetT, ΔputPA, ΔproP, and ΔproU)to test for the accumulation of osmolytes and salt stress tolerance. (2). transform and over-express each osmolytes synthesizing gene in E. coli BL21 (DE3)-RIL and purified protein for enzyme activity assay and further structure analysis. (3). transform and over-express osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately into E. coli BL21 (DE3)-RIL and tested for intracellular osmolyte accumulation and salt/temperature stress tolerance. (4). transform and over-express osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately into Corynebacterium glutamicum for osmolytes production. These osmolytes can be used for salt, chilling, drought protectants and humectants for food and agriculture industries. (5). transformed and over-expressed osmolytes betaine, Nε-acetyl-β-lysine and β-glutamine synthesizing gene sets separately to Arabidopsis thaliana and Zebrafish and tested for intracellular osmolyte accumulation and salt/ temperature stress tolerance. The study in methanogenic archaea, the widest salt growth range of living organism, at salt stress-response should gain the basic knowledge in osmoregulation and application in anti-salt and drought organisms and macromolecules

台灣本土甲烷太古生物的收集、特性分析、系統演化分類鑑定與應用潛力分析(I)

此研究計畫期藉由多樣化物種的收集探討，提供生物科技發展的資源。最新的生物分類法利用rRNA 基因和分子生理特性將生物界重新分成『ThreeDomains』-Archaea, Eubacteria 及Eukarya。 其中，俗稱第三類生物的太古生物(Archaea)兼具原核與真核的特色，且大多為生長在極端高溫、高鹽或無氧的極端環境中的單細胞原核生物。 由於極端環境生物的培養較為困難，因此國內在這方面的研究探討甚為稀少，對本土這方面的資源開發也遠落後於西方先進國家。 絕對厭氧的甲烷太古生物(以下簡稱甲烷菌)存在於任何無氧、低氧化還原電位的環境，利用簡單的H2/CO2，formate，acetate，methanol，及methylamines 作為碳源進行甲烷化作用(methanogenesis)，產生甲烷，提供能源和大氣中碳的循環並在生物的食物鏈中佔有相當重要的地位。 甲烷菌是太古生物中唯一能適應多變化的環境如溫度、鹽度和酸鹼值的生物，不僅能分佈於高溫(100 ) ℃ 及低溫(2 ) ℃ 的環境，也能生長於相當廣泛的不同鹽濃度下(0-4.3 M NaCl)。 計劃申請人實驗室七年前開始在本土自然環境收集純化甲烷菌，目前已純化21 株甲烷菌，分別寄存於美國和德國的菌種中心，且已完成16 株菌的16S rRNA 基因序列的定序與比對。 其中，有三株已確定為新種， Methanofollis aquaemaris ， Methanocalculus taiwanensis 及Methanocalculus chunghsingensis。我們的研究發現，自本土人為環境中收集的甲烷菌多和國外已發表菌株相似，但由自然環境純化的多為新種或新屬。因此在四年期計劃中，我們規劃自台灣的自然環境如地熱區、泥火山、紅樹林等處取樣，純化收集更多的甲烷菌並計劃完成全部我們已純化的21 株自台灣本土純化的甲烷菌之分類鑑定。另外，我們純化的Methanosarcina 對氧氣和較高的氧化還原電位有耐受性，適用於兼性厭氧環境的處理槽；我們純化的新種Methanocalculus chunghsingensis具有聚磷能力，可用以除磷；將分析其特性並嘗試開發應用於環保生技。此外，甲烷菌型軟骨素與表層蛋白可開發作為生物材料

Characterization and Application of Osmolyte Synthetic Genes of Methanogenic Archaea

極端厭氧的甲烷太古生物 (Archaea, 又稱古菌、古生菌或古細菌)，以甲烷的生合成方式提供細胞所需的能量，能生存於各式溫度(2- 117 ℃)、酸鹼度(中性至強鹼)及鹽度(淡水至飽合鹽度)等環境，是唯一能適應多變化環境的太古生物。我們實驗室收集國內外能生長在各種不同鹽度的甲烷菌，長期探討甲烷菌對鹽逆境的反應與適應機制。我們的研究顯示嗜鹽甲烷菌具有一高親和性、高專一性，需能且受滲透壓調控的ABC型式的甜菜鹼運輸系統Bta，會自胞外環境優先攝取相容質甜菜鹼。而當胞外環境缺乏相容質的提供時，能自體生合成相容質β-glutamine、Nε-acetyl-β-lysine及甜菜鹼 (betaine)於細胞體內，以維持膨壓及保護胞內蛋白等大分子。除了利用各種特殊相容質的累積來適應環境中的不同鹽度之外，我們最近的研究首次發現分子伴蛋白chaperone ClpB亦參與鹽逆境的反應，在高鹽、低鹽逆境的表現量會增加，且會受相容質甜菜鹼的抑制。為探討相容質與甲烷古菌對鹽逆境的適應機制，我們目前已獲得嗜鹽甲烷菌Methanohalophilus portucalensis FDF1相容質betaine的生合成酵素GSMT與SDMT的基因。在甲烷菌的共通相容質Nε-acetyl-β-lysine生合成基因部分，我們已分別獲得海洋甲烷菌Methanosarcina mazei N2M9705、耐鹽甲烷菌Methanocalculus chunghsingensis K1F9705b與嗜鹽甲烷菌Methanohalophilus portucalensis FDF1的lysine 2,3-aminomutase (ablA)與β-lysine acetyltransferase (ablB)基因；以及淡水型甲烷菌M. mazei N2M9705的相容質β-glutamine 的生合成基因。在此三年期計畫期間，將分析甲烷菌相容質生合成基因的特性與系統演化分析；探討不同鹽度、溫度或相容質betaine 等因子對甲烷菌自體生合成的相容質基因之轉錄表現的影響與調控。此外，將相容質生合成相關基因分別轉殖到大腸桿菌大量表現並純化蛋白進行酵素活性分析；並將生合成相容質β-glutamine、Nε-acetyl-β-lysine及betaine的基因組，分別轉殖到大腸桿菌與阿拉伯芥上，分析其相容質生成累積量與探討是否能提高其對鹽度的耐受性。相容質與滲透壓的基礎與應用研究，從抗鹽相容質glycerol、trehalose和近年發展的高鹽細菌生產的ectoine在發酵工業、食品界、化妝美容界、製藥與醫療等的應用，可以想見微生物因應鹽與滲透壓力的小分子相容質在基礎研究與應用的重要性。絕對厭氧的甲烷太古生物能生長在淡水至飽和鹽環境，對鹽逆境與滲透壓逆境的適應是生物界中最寬廣的，期由對甲烷太古生物在鹽逆境的反應與適應機制相關基因與調控的瞭解及應用潛力的分析，進而提高生物抗鹽抗旱的能力

Construct the Taiwan Archaeal Biota and Investigate the Taiwan Ddepsea Prokaryotic Diversity

本研究將(1). 加強分類學能力建設，逐年完成台灣微生物誌之編撰。我們過去在農委會與國科會的部分支持下，在彰化二林溪出水口、淡水河域與墾丁海域附近收集純化甲烷太古生物，純化鑑定三十六株，其中四株已確定且發表為新種，Methanofollis aquaemaris、Methanofollis formosanus、Methanocalculus taiwanensis及Methanocalculus chunghsingensis。我們也由「台灣車籠埔斷層深井鑽探計畫」於大坑的鑽探的深井取樣，純化出取自地表底下545 m岩心樣品純化Methanolobus chelungpuianus St545MbT and 自地表底下694 m 岩心樣品的Methanobacterium palustre FG694aF。此外，我們也自苗栗通宵的鹽山上收集純化十八株特殊方型或三角形菌體的極端高鹽太古生物Haloarcula屬與Natrinema屬菌株。將陸續將這些本土太古生物的分類鑑定資料，依台灣生物多樣性資訊網(TaiBNET)之生物誌(Biota Taiwanica)規範與Bergey’s Manual of systematic Bacteriology 規範建議撰寫中英文生物誌。(2). 我們規劃進行臺灣深海原核生物多樣性探討。過去三年參與經濟部地質調查所委辦台灣西南海域新興能源-天然氣水合物資源調查與評估計畫，藉由海研一號及三號及法國研究船Marion Dufresne，我們自台灣西南海域取得超過百個深海岩心不同深度底泥的樣品，我們目前已經分析超過2400個地質微生物(細菌與太古生物)16S rRNA基因，經由生物資訊分析與資料庫比對，發現這些樣點的微生物和目前已知的菌種相似度低，顯示台灣西南海域深海微生物的驚人多樣性，是重要的微生物資源寶庫。希望能進一步純化鑑定我們自西南海域甲烷水合物區的樣品中之原核生物(細菌與太古生物)。這些台灣深海微生物菌株之多樣性資源，可提供台灣海域微生物物種與基因的多樣性資料；將來可推動發展應用本土生物的生物技術，促進本土生物資源的永續應用。In this grant proposal, first, we would like to write the Taiwan microbial biota related to methanogenic archaea and extreme halophilic archaea isolated from Taiwan. With the partial grant support from Council of Agriculture and National Science Council, we have isolated and characterized 36 strains of methanogenic archaea from Estuarine in Eriln Shi, Kaomei wetland, Aquaculture fishpond in Wang-gong, Danshuei River, Kenting and Anaerobic Digester. Among them, four strains have been identified and characterized as new species and they are Methanofollis aquaemaris, Methanofollis formosanus, Methanocalculus taiwanensis and Methanocalculus chunghsingensis. Additionally, new methanogen isolates, Methanolobus chelungpuianus St545MbT and Methanobacterium palustre FG694aF, were isolated from core samples obtained from deep subsurface of the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan at 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 meter below land surface (mbls). Also, 18 strains of extreme halophilic archaea, Haloarcula and Natrinema, were isolated and characterized from the salt crystals obtained from the salt mountain at Miaoli, Taiwan. We will gradually compose above Taiwan local archaeal information as biota format according to the TaiBNET requested format. Second, we would like to investigate the diversity of deep sea Prokaryotes. During the past three years, we joined the project initiated and supported by Central Geological Survey in methane hydrate energy investigation off shore of Taiwan. From core drilling cruises by Ocean Research I, Ocean Research III and RV Marion Dufresne, we have obtained more than 100 sediment samples from various depths of cores and more than 2400 clones of 16S rRNA gene sequence of archaea and bacteria were analyzed. Through the database comparison and bioinformatic analysis, the16S rDNA sequences of all clones slowed low similarity with known cultured organisms, which indicated these are all unknown living organisms and these sites can be great pool for bio-resources. We would like to further isolate, characterized these deep sea prokaryotic organisms. These investigations should enhance our understanding in our marine microbial diversity and should further supply the species, genetic information for further biotechnological application